Voltage-dependence of ciliary activity in the ciliate Didinium nasutum

Ciliary organelles serve a multiplicity of physiological functions in widely different phylogenetic systems. Besides their function in feeding (Ciliata: Fenchel, 1980; Placozoa: Wenderoth, 1990; Mollusca: Stommel and Stephens, 1988), sensory perception (Vertebrata: Frings and Lindemann, 1991) and transport (Vertebrata: Sanderson et al. 1992), locomotion is the main function of ciliary performance (Ciliata: Machemer, 1974; Ctenophora: Mogami et al. 1991; sperm cells: Okuno and Brokaw, 1981).

Ciliary activity is due to microtubular sliding of the axonemal complex (Satir, 1989). The axoneme consists of the highly conserved 9+2 pattern of microtubules (Witman, 1990) with dynein as the force-generating ATPase (Warner, 1989). Relative sliding between peripheral microtubular doublets produces cyclical bending of the axoneme (Holwill, 1989). This movement generates the propulsive force for locomotion and is the cause of water streaming around the cell (Sleigh, 1991).

In ciliates, axonemal activity and, therefore, swimming behaviour are controlled by the membrane potential (Machemer, 1989, 1990). Electromotor coupling of cilia is also found in ctenophores (Moss and Tamm, 1986), molluscs (Murakami, 1968) and vertebrates (Boitano and Omoto, 1991). Basic experiments concerning electromotor coupling in ciliates were performed using Paramecium caudatum (Kinosita et al. 1964) and Euplotes sp. (Naitoh and Eckert, 1969) and, more recently, these have been extended to other ciliates such as Stylonychia mytilus (Deitmer et al. 1984) and Blepharisma japonicum (Matsuoka et al. 1991).

The free Ca²⁺ concentration plays a crucial part as an intracellular messenger in electromotor coupling (Mogami et al. 1990). Extracellular Ca²⁺ enters the cell because of changes in the conductance of voltage-activated Ca²⁺ channels (Pernberg and Machemer, 1989), thus increasing the intracellular Ca²⁺ concentration (J. Pernberg and H. Machemer, in preparation).

Didinium nasutum offers considerable advantages for analyzing ciliary activity since the restriction of the cilia to two circular bands (Lipscombe and Riordan, 1992) and good metachronal coordination aid identification of the beating mode of cilia.

After extensive electrophysiological characterization of Didinium nasutum (Pape and Machemer, 1986; Pernberg and Machemer, 1989), this work analyzes the voltage-dependent ciliary activity in relation to inactivation. Compared with Paramecium caudatum and other ciliates, Didinium nasutum exhibits some interesting differences in ciliary beating mode. Our results enable us to illuminate further the role of free Ca²⁺ as an intracellular messenger in electromotor coupling.

Didinium nasutum was cultured in Pringsheim solution at 18 °C and fed with Paramecium caudatum every second day. Egg-shaped Didinium nasutum had a mean length of 127 μm (93–154 μm) with a maximum diameter of 80 μm (57–95 μm). Cells were ready for experimentation 20 h after feeding.

For recording of ciliary beating, a high-frequency video system (NAC, Tokyo) was used. Image frequency was 200 Hz and a single flash of stroboscobic illumination lasted 15 μs. During the experiments, a specimen was voltage-clamped at a holding potential equal to the resting potential (approximately -35 mV). The cell was under optical control using a standard microscope equipped with a differential interference contrast device (Zeiss, Oberkochen).

The direction of the cilium at the end of the effective stroke with respect to the longitudinal axis of the cell was defined as the beating angle. At this position, cilia showed a typical straight configuration. To analyze the beating angle, ciliary activity was documented as viewed from above. With the cell soma in the background, the contrast of the cilia decreased when compared with the profile view used for the acquisition of frequency data. For this reason, only a few video sequences could be analyzed in terms of beating angle owing to the critical dimensions of a single cilium (diameter approximately 0.25 μm).

Cells were equilibrated for more than 1 h in the experimental solution of 1 mmol l^-1 KCl, 1 mmol l^-1 CaCl₂ and 1 mmol l^-1 Tris buffer at pH 7.2–7.4. Ciliary beating patterns were studied during the application of 450 ms voltage steps to different step potentials. Room and bath temperatures were kept constant at 18–20 °C. For electrophysiological details, see Pernberg and Machemer (1989).

Each of the approximately 3000 cilia (Pape and Machemer, 1986; estimated after Wessenberg and Antipa, 1968) is 17 μm long and has a diameter of 0.25 μm. They are densely packed in the anterior and equatorial belts. Beating frequency and the direction of the effective stroke are conventional parameters with which to describe the cyclic ciliary activity.

Didinium nasutum swam in a right-handed helix with a velocity of 1.6±0.1 mm s^-1 (continuous forward swimming; mean ± S.D.; N=20 cells). Spontaneous reversals were observed at mean intervals of 2.9±1.0 s (N=38). The duration of backward swimming was very short (175±61 ms; N=26). This value corresponds to the duration of spontaneous ciliary reversals (177±80 ms; N=9).

In a tip-to-base view, the cilium moved in a counterclockwise direction for all observed beating patterns. Each ciliary cycle was seen to have a clear polarity. During the force-generating effective stroke, the cilium was bent at its base and the tip shifted at the maximum distance from the cell surface. During the return stroke, the entire cilium moved close to the cell surface and was bent more or less throughout.

We define ciliary activity under resting conditions as the ciliary movement observed at a membrane holding potential (=resting potential) of -35±3 mV (N=20 cells). The resting frequency of the cilia was 14.7±2.6 Hz. This value was calculated for each cell within an 800 ms prestimulus time. As long as the holding potential was unchanged, the beating frequency was constant (Fig. 1) and varied from 10 to 20 Hz in individual cells. The direction of the effective stroke pointed posteriorly and to the left at an angle of 18 ° to the longitudinal cell axis (see below). Metachronism was dexioplectic; that is, with the effective stroke pointing posteriorly and to the left, the metachronal wave moved in a 90 ° counterclockwise direction (see Fig. 4C, inset top).

During hyperpolarizing voltage steps with a maximal amplitude of -35 mV, no change of the beating mode was detected (Figs 1A, 2A). Depolarizations of more than 5 mV increased the beating frequency from the resting value (14.7 Hz) to 26.0±2.4 Hz (N=22 cells). The directions of both the effective stroke and the metachronal wave reversed (see Fig. 4C, insets). The dexioplectic metachronal coordination persisted. After repolarization to the resting potential, ciliary beating frequency returned to the resting value. With increased voltage steps, the duration of reversed ciliary beating after the end of the voltage step increased, but rarely exceeded 0.5 s (Fig. 1B,C).

To analyze the voltage-dependence of ciliary beating, the change in frequency was plotted against the membrane potential. The change in frequency (Δfrequency) was calculated by subtracting the resting frequency from the mean frequency during the step potential (Fig. 2A). We defined as the ‘physiological voltage range’ all membrane potentials between -70 mV and 0 mV. Potentials measured in the ‘free’, unclamped cell are within this range when measured, for instance, during spontaneous action potentials (Pernberg and Machemer, 1989).

Increasing the negativity of the membrane potential changed neither the frequency nor the direction of ciliary beating (Fig. 2A). Thus, in contrast to Paramecium caudatum (Machemer, 1988) and Stylonychia mytilus (Mogami and Machemer, 1991), a hyperpolarization-induced ciliary activation (HCA) is missing in Didinium nasutum. At membrane potentials more positive than -25 mV (Δ=+10 mV), reversed beating occurred (depolarization-induced ciliary activation, DCA). Within the voltage range -25 mV to 0 mV, the frequency and direction of reversed beating were unchanged.

Near the ‘transition potential’ of -30 mV (Δ=+5 mV), cilia on some cells still showed normal beating, cilia on others beat in the reverse direction, while a third group of cilia was inactivated (Fig. 2). For each cell, inactivation was seen within a voltage range of a few millivolts (Fig. 2B). With decreasing negativity of the membrane, the proportion of cells that had undergone inactivation before changing to reversed beating increased. During the phase of inactivation, the cilia assumed a more or less straight conformation virtually perpendicular to the cell surface.

Ciliary inactivation always occurred during the relatively slow transition from normal to reversed beating (or vice versa, Fig. 3). A small depolarizing stimulus, which was insufficient to evoke reversed beating, tended to inactivate the cilia (Fig. 3A). With a larger step amplitude, reversed beating occurred at the end of the voltage step after a period of ciliary inactivation (Fig. 3B). A second ciliary inactivation preceded the restoration of normal beating. At depolarizations positive to the transition potential of -30 mV, inactivation typically lasted 200–500 ms during the transition from reversed to normal beating (Fig. 3C). At these potentials, the transition from normal to reversed beating at the onset of the depolarization was rapid, suggesting the total absence of a transient state of ciliary inactivation.

Extreme membrane potentials, beyond the physiological voltage range, were applied to manipulate the driving force for Ca²⁺ (Fig. 4). A second transition potential was seen at positive voltage steps to +65 mV. Near this potential, ciliary reversal tended to transform to normal beating of the cilia. With even more positive voltages, no reversed beating occurred; that is, neither the frequency nor the direction of beating was changed (Fig. 4A). A brief reversal was seen, however, after the end of the voltage step (an off-response, Fig. 4B).

During negative potentials between about -100 mV and -200 mV, normal beating, reversed beating and ciliary inactivation were observed. With even more negative potentials, the proportion of events of reversed beating increased. In the reversed cilia, the increment in frequency rose as a linear function of voltage (with a slope of approximately 7 Hz per 100 mV; line in Fig. 4A). The frequency saturated at more negative potentials. The maximal frequency corresponded to that seen during depolarizations.

At the resting membrane potential (-35 mV), the power stroke of the cilium was posteriad; that is, 18 ° to the left of the longitudinal axis of the cell (198 ° in Fig. 4C). Within the physiological range of hyperpolarizing voltage, no change in beating angle was detected. Whenever, under extreme negative and positive potential steps, the direction of beating changed from normal to reversed, the resulting power stroke was anteriad 24 ° to the right of the longitudinal axis (Fig. 4C). This corresponds to a ciliary reversal in the counterclockwise direction of 174 °.

The three-dimensional ciliary cycle of Didinium nasutum, as well as that in Paramecium caudatum (Machemer, 1972) and Stylonychia mytilus (Machemer and Sugino, 1986), describes an imaginary cone. The effective stroke and the return stroke are distinct both temporally and spatially (Sugino and Naitoh, 1982). The dexioplectic metachronism seen in Didinium nasutum (Fig. 4C) is common to all ciliates so far investigated (Machemer, 1974) and has also been found in the Metazoa (Knight-Jones, 1951).

With a mean swimming speed of 1.6 mm s^-1, Didinium nasutum moves faster than Paramecium caudatum (0.8–1.2 mm s^-1, Machemer, 1989). Both the duration (100 ms, Machemer, 1989) and the frequency (one reversal per 5–10 s; Becker, 1983) of spontaneous reversals of Paramecium cilia are smaller than those in Didinium nasutum (175 ms, one reversal per 2.9 s).

Spontaneous action potentials, inducing reversals in forward swimming, are caused by fluctuations in the conductance of ion channels within the cell membrane (Martinac et al. 1986). They have been observed in various ciliates (Naitoh, 1966; Kokina, 1970; Moolenaar et al. 1976; Deitmer et al. 1986) and in Opalina ranarum (Kokina, 1964).

Within the physiological voltage range, a hyperpolarization-induced ciliary activation (HCA) was missing in Didinium nasutum which, therefore, differs from Paramecium caudatum and Stylonychia mytilus (De Peyer and Machemer, 1982b; Machemer, 1986). Depolarization-induced ciliary activation (DCA), in contrast, resembles the DCA seen in these two ciliates (Machemer and De Peyer, 1982; Mogami et al. 1990). The intraciliary Ca²⁺ concentration is believed to be the messenger regulating DCA, as documented by in vitro reactivation experiments in Paramecium caudatum (Naitoh and Kaneko, 1972; Nakaoka et al. 1984). The elevation of the ciliary Ca²⁺ concentration results from a voltage-gated influx of Ca²⁺ from the extracellular milieu.

The gradual decrease in beating frequency after the end of the depolarizing voltage step (Fig. 1B) corresponds to the kinetics by which the elevated Ca²⁺ concentration is believed to return towards the resting value. Removal of Ca²⁺ is mediated by an active transport mechanism (Hildebrand, 1978), presumably an ATPase which pumps Ca²⁺ from the ciliary cytoplasm (Noguchi et al. 1979; Martinac and Hildebrand, 1981). This assumption is indirectly supported by experiments showing that a reduction in temperature results in a counterclockwise shift of the effective ciliary beat in Paramecium caudatum corresponding to an increase of the intraciliary Ca²⁺ concentration (Machemer, 1972). Moreover, an inhibition of the presumed active transport system extended the duration of DCA (Doughty, 1978; Hildebrand, 1978).

Following the same line of reasoning, the extension of DCA with increasingly depolarizing voltage steps (Fig. 1B,C) results from a more elevated Ca²⁺ concentration and a longer period of Ca²⁺ pumping (De Peyer and Machemer, 1982a; Mogami and Machemer, 1991).

Ciliary inactivation is seen in various ciliates when a small increase in the ciliary Ca²⁺ concentration takes place. In Paramecium caudatum, transient ciliary inactivation was observed after a depolarization during the transition from reversed to normal beating (Machemer, 1974, 1975). In Stylonychia mytilus, the marginal cirri are inactive at the resting potential. This intermediate mode between normal and reversed beating is associated with the resting potential (De Peyer and Machemer, 1983).

In conclusion, the electromotor coupling and cell reactivation data from different ciliate species show that, within a narrow range of slightly raised intraciliary Ca2+ concentrations, cyclic ciliary beating is suppressed. Although the mechanism is unknown, inactivation may be important for ciliary reorientation during the change in beating direction. A raised intraciliary free Ca²⁺ concentration during inactivation and reversed beating may affect the axonemal complex directly or via calmodulin (see Preston and Saimi, 1990). Rates of active sliding and sliding translocation between microtubule doublets (Sugino and Naitoh, 1982; Sugino and Machemer, 1988) and/or the dynein mechanochemical cycle (see Omoto, 1991) could be changed. Mogami and Machemer (1990) presented a quantitative model that included a modulatory function for Mg²⁺, which competes with Ca²⁺ for binding to an axonemal protein.

Ca²⁺ influx through the ciliary membrane depends on (1) the conductance for Ca²⁺ and (2) the driving force for Ca²⁺. Augmentation of each of these leads to an elevated Ca²⁺ concentration. Depolarizing voltage steps increase the Ca²⁺ conductance; very large positive steps depress, in addition, the driving force for Ca²⁺. Extreme hyperpolarizations, however, increase the driving force.

With the ciliary resting conductance unchanged during hyperpolarizations, Ca²⁺ influx is slightly augmented as a result of the increased driving force for Ca²⁺. The gradual augmentation in inward leakage of Ca²⁺, and hence of the intraciliary Ca²⁺ concentration, explains the gradual increase in the frequency of reversed beating (line in Fig. 4A). Thus, it is possible that there is a graded ciliary frequency response in Didinium nasutum, but it is not seen under physiological stimulus conditions because the corresponding range of voltage-regulated conductance is very narrow. The gradual frequency increase was documented for hyperpolarizations up to -200 mV, corresponding to a doubling of the driving force and Ca²⁺ entry [Ca²⁺ equilibrium potential (E_Ca)=+107 mV; J. Pernberg and H. Machemer, in preparation]. From this, it can be inferred that, if depolarizations in 1 mV increments induce a transition from normal to reversed beating at maximal frequency, the ciliary Ca²⁺ conductance was at least doubled.

Stepwise rising depolarizations did not result in graded ciliary responses (Fig. 2A). Near the transition potential of -30 mV, reversed beating was associated with a maximal frequency increase. In order to identify a possible graded ciliary motor response in this voltage range, depolarization step intervals should be smaller than 1 mV. This kind of experiment has not yet been performed.

With extreme depolarizing membrane potentials of +65 mV or greater, DCA was no longer elicited. This suggests that, beyond this transition potential of +65 mV, the entrance of Ca²⁺ through voltage-activated Ca²⁺ channels was insufficient to elevate the intraciliary Ca²⁺ concentration above the threshold for DCA. After stepping back to the holding potential (-35 mV), Ca²⁺ influx through still open Ca²⁺ channels elicited a ciliary ‘off-response’ (Fig. 4B).

A voltage-dependent suppression of DCA at very positive membrane potentials has been reported in other species of ciliates (Paramecium caudatum, +70 mV, Machemer and Eckert, 1975; Stylonychia mytilus, +100 mV, De Peyer and Machemer, 1982a; +104 mV, Mogami and Machemer, 1991). In Didinium nasutum, the Ca²⁺ equilibrium potential is near +107 mV (external [Ca²⁺], 10⁻³ mol l^-1; internal [Ca²⁺], 2 ×10⁻⁷ mol l^-1, J. Pernberg and H. Machemer, in preparation). A marked discrepancy (about 40 mV) exists between the observed transition potential (+65 mV) and the calculated value for E_Ca. A possible explanation is that the Ca²⁺ influx is too small to increase the Ca²⁺ concentration above the threshold for DCA. This could only be true for a minor conductance of Ca²⁺, which is unlikely because the open probability of voltage-dependent Ca²⁺ channels increases to a maximum as the membrane potential becomes more positive (Chen and Hess, 1990; Pietrobon and Hess, 1990).

The reversal potential for the early inward current (+45 mV; Pernberg and Machemer, 1989) also differs from the value of E_Ca. It might, therefore, be argued that an equilibrium potential of +107 mV does not apply to the Ca²⁺ channels in the ciliary membrane. This paradox may be explained by assuming a screening effect on the ciliary Ca²⁺ channel. Positive charges next to the external mouth of the channel could repel Ca²⁺ electrostatically, and such screening could be due to the presence of free or bound cations.

Screening could explain why a reduction of the early Ca²⁺ influx is found when the total concentration of extracellular cations is increased (Oka and Nakaoka, 1989). With the hypothetical assumption of an effective Ca²⁺ equilibrium potential of +65 mV across the Ca²⁺ channel, the concentration of free Ca²⁺ at the outer face of the channel would be 3.5 ×10⁻⁵ mol l^-1 (3 % of the nominal value of 1 mmol l^-1).

At a depolarization of 10 mV, reversed beating was seen on the first video frame after the onset of the voltage step; that is, after less than 5 ms. During this time, the maximal Ca²⁺ influx is 10⁻¹⁷ mol (Pernberg and Machemer, 1989). The total volume of all cilia in Didinium nasutum is 2.5 ×10⁻¹² l. Assuming that the volume of the ciliary liquid space is 50 % of the geometric volume (Machemer, 1986), the Ca²⁺ influx results in an increase in intraciliary Ca²⁺ concentration of up to 8.5 ×10⁻⁶ mol l^-1. Using this concentration together with the latency time and the driving force for Ca²⁺, a minimal estimate of the resting resistance of the ciliary membrane can be obtained. Applying Ohm’s law, the quotient of the difference between driving force and membrane potential (voltage), on the one hand, and the charge influx per time (current), on the other hand, gives the resistance. Data from extreme hyperpolarizations in 13 cells suggest a minimal resting resistance of 10 GΩ. Using a total ciliary surface of 4 ×10⁻⁴ cm² (Pape and Machemer, 1986), the minimal specific resting resistance is 4 ×10⁶ Ω cm².

The limited temporal resolution of the video system (5 ms, see Materials and methods) means that the calculated value for the intracellular [Ca²⁺] threshold of ciliary reversal (8.5 ×10⁻⁶ mol l^-1) is an overestimate. Thus, the estimate of the resting resistance of the ciliary membrane of 10 G Ω is on the safe side. Assuming an intracellular [Ca²⁺] threshold for ciliary reversal of 6 ×10⁻⁷ mol l^-1, as determined from reactivated models of Paramecium caudatum (Nakaoka et al. 1984; Noguchi et al. 1991), the ciliary resting resistance is 200 G Ω (specific resistance 8 ×10⁷ Ω cm²). In agreement with this, deciliation experiments in Paramecium caudatum (Machemer and Ogura, 1979) and Didinium nasutum (Pape and Machemer, 1986) suggested an extremely high ciliary membrane resistance because deciliation, that is removal of 50 % of the total cell surface, did not raise the input resistance of the cells.

Voltage-dependent Ca²⁺ channels from the ciliary membrane of Paramecium caudatum placed into lipid bilayers gave a single-channel conductance of 1.5–2.0 pS (Ehrlich et al. 1984). This value is smaller than that for mammalian cells (3–25 pS, Carbone and Lux, 1987; Fox et al. 1987; Droogmans and Nilius, 1989). Assuming that Didinium nasutum has a similiar channel conductance to the ciliary Ca²⁺ channel suggested for Paramecium caudatum, a ciliary resting resistance of 200 G Ω would result from the opening of 2–3 Ca²⁺ channels. At 0 mV, the ciliary resistance is minimally 21 M Ω (Pernberg and Machemer, 1989). This corresponds to a ciliary conductance of 4.8 ×10⁻⁸ S or 3 ×10⁴ open Ca²⁺ channels. If all voltage-dependent ciliary Ca²⁺ channels are open at this potential and if they are equally distributed over all 3000 cilia, a ciliary membrane would incorporate 10 fast-activating Ca²⁺ channels. Similar values for the resistance of the depolarized ciliary membrane have been calculated in Paramecium caudatum (2.5 ×10⁻⁸ S, Schein et al. 1976; 2.7 ×10⁻⁸ S, Oertel et al. 1977). In Didinium nasutum, an activated ciliary membrane conductance of 1.6 ×10⁻¹¹ S corresponds to previous estimations for Paramecium caudatum (1.7 ×10⁻¹¹ S, Eckert and Brehm, 1979). With an intraciliary resting Ca²⁺ concentration of 2 ×10⁻⁷ mol l^-1 (J. Pernberg and H. Machemer, in preparation), the number of calcium ions within a single cilium must rise by 1.7 ×10⁻²² mol (approximately 100 ions) to reach the threshold for reversed beating.

According to the ‘two-threshold’ Ca²⁺ hypothesis (Eckert, 1972), an increase in the driving force during membrane hyperpolarization increases the inward Ca²⁺ leakage. From this, the intraciliary Ca²⁺ concentration can reach the threshold for augmentation of the beating frequency seen during hyperpolarization in Paramecium caudatum. Moreover, a reversal in beating direction should occur when the Ca²⁺ concentration passes a second, even higher, Ca²⁺ concentration threshold as a result of further elevation of the driving force for Ca²⁺. In Didinium nasutum, a hyperpolarization-induced increase of the Ca²⁺ leakage current and intraciliary Ca²⁺ concentration results in a frequency augmentation of only those cilia beating in reverse. A voltage-dependent, gradual increase of the beating frequency was seen exclusively in these cilia. Therefore, it has to be concluded that there is a single Ca²⁺ threshold for both frequency increase and reversal of beat direction. Individual differences in threshold voltage for reversed beating can be explained by assuming a divergence in resting Ca²⁺ conductances.

How do Ca²⁺ conductances (Pernberg and Machemer, 1989) regulate the Ca²⁺-dependent ciliary movement and, consequently, the locomotion of Didinium nasutum? The fast transient Ca²⁺ conductance generates the regenerative depolarization, a rapid rise in the Ca²⁺ concentration and ciliary reorientation (reversed beating) together with an increased beating frequency. This relationship corresponds to data from other ciliates (Machemer and Eckert, 1975; De Peyer and Machemer, 1982a; Machemer and Sugino, 1986).

A later-activating, persistent Ca²⁺ conductance supports continued ciliary beating in reverse. As discussed for Paramecium caudatum (Machemer and Eckert, 1975), a slowly inactivating Ca²⁺ influx is responsible for intraciliary Ca²⁺ concentrations above the threshold for ciliary reversal. A late increase in K⁺ conductance supports membrane repolarization and, therefore, deactivation of the Ca²⁺ influx. After substitution of Ba²⁺ for Ca²⁺, the inactivation of the early inward current and the late K⁺ efflux were inhibited, resulting in a widening of the action potentials (Didinium nasutum, Pernberg and Machemer, 1989; Paramecium caudatum, Brehm et al. 1978).

In Didinium nasutum, maintenance of the resting membrane potential at a constant level allows steady ciliary beating and forward swimming of the cell. Transient backward swimming results primarily from spontaneous action potentials. The repeated reorientations that follow such transient reversals may increase the probability of encounters with prey cells (such as Paramecium caudatum).

The levels of membrane depolarization normally achieved fall because of the Ca²⁺-and voltage-dependent inactivation of the Ca²⁺ channels. Only when more positive membrane potentials are reached does the late-activating K⁺ conductance repolarize the membrane. The major function of this conductance is, therefore, the homoeostasis of the cell membrane.

We are grateful to Dr G. Schweigart for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft, Forschergruppe Konzell 3.